HYALURONAN AND EPIDERMAL DIFFERENTIATION

描述(由申请人提供)： 我们的总体目标是验证一种假说，即透明质酸(透明质酸，HA)是皮肤角质形成细胞之间细胞间隙中的一种丰富的基质成分，在表皮形成和创面愈合过程中起着至关重要的作用，并参与协调角质形成细胞增殖和分化的机制。我们的初步数据表明，HA不是简单的细胞外空间的被动占有者，而是在调节表皮分化程序中具有积极的指导作用。使用专门为这些研究量身定做的体外模型(在RAFT培养中分层的大鼠角质形成细胞系)，我们将检测表皮HA的代谢及其在调节表皮分化中的功能作用。目的1对角质形成细胞在分化过程中产生的透明质酸、主要的透明质酸合成酶的表达以及透明质酸酶的表达进行量化。将使用链霉菌透明质酸酶试验性地将HA从细胞间隙中移除，以创建一种“功能性HA敲除”，在这种敲除中可以评估对分化和增殖的影响。目的2将更仔细地探索HA代谢的合成方面，使用Tet-诱导型(Teton)基因开关系统在表皮分化的不同时间在角质形成细胞中过表达HAS2。Aim 3将在分解代谢方面采取类似的方法，通过诱导过度表达透明质酸2，这是在REK细胞中表达和调节的主要透明质酸酶。Hyal2将过度表达，并确定HA降解增加的后果。在相关实验中，CD44(角质形成细胞质膜上的主要透明质酸受体，与HA的摄取和降解有关)将成为靶点。缺乏细胞质锚定尾巴的显性-阴性形式的CD44将被诱导过度表达，从而干扰天然的CD44结合和功能。Aim 4将把从先前的AIMS中获得的信息应用到体内无毛小鼠皮肤的实验中。利用两种常见的表皮增生模型，即(I)局部应用维甲酸，以及(Ii)破坏角质层的屏障，我们将确定HA是否在对表皮特异性刺激和损伤的反应中起作用。

DESCRIPTION (provided by applicant): Our overall goal is to test the hypothesis that hyaluronan (hyaluronic acid, HA), an abundant matrix component in the intercellular space between keratinocytes of the skin, has a crucial role in epidermal homeostasis and participates in a mechanism to coordinate keratinocyte proliferation and differentiation during epidermal formation and wound-healing. Our preliminary data indicate that HA is not simply a passive occupant of the extracellular space, but rather has an active, instructive role in regulating the epidermal differentiation program. Using an in vitro model specifically tailored for these studies (a rat keratinocyte line that stratifies in raft culture), we will examine epidermal HA metabolism and its functional role in regulating epidermal differentiation. Aim 1 will quantify the production of HA, expression of the major HA synthase enzymes, and expression of hyaluronidases produced by the keratinocytes during differentiation. HA will be experimentally removed from the intercellular space using Streptomyces hyaluronidase, to create a "functional HA-knockout," in which effects on differentiation and proliferation can be assessed. Aim 2 will more-carefully explore the synthetic side of HA metabolism, using a tet-inducible (tetON) gene-switch system to overexpress HAS2 in the keratinocytes at different times during epidermal differentiation. Aim 3 will take a similar approach on the catabolic side, by inducibly overexpressing HYAL2, the major hyaluronidase expressed and regulated in the REK cells. HYAL2 will be overexpressed, and the consequences of increased HA degradation determined. In related experiments, CD44 (the major hyaluronan-receptor on the keratinocyte plasma membrane, and implicated in the uptake and degradation of HA) will be targeted. A dominant-negative form of CD44 that lacks the cytoplasmic anchoring tail will be inducibly overexpressed to interfere with native CD44 binding and function. Aim 4 will apply information gained from the prior Aims to experiments in hairless mouse skin in vivo. Using two well-known models of epidermal hyperplasia, namely (i) application of topical retinoids, and ii) barrier disruption of the stratum corneum, we will determine whether HA has a role in responses to epidermal-specific irritation and injury.